Quantitative Analysis of NAD+ and NAM

NAD⁺ and NAM extraction and subsequent quantitative analysis were performed as previously described (Yoshino and Imai, 2013 (link)). Briefly, tissue sample (typically 50–70 mg) was homogenized in 500 µL HClO₄ (0.4 M), incubated on ice for 5 min and precipitated by adding 80 µL KOH (0.2 M) with shaking for 2 min. The samples were then centrifuged at 3000 × g, 10 min, 4°C. Supernatant was filtered using Spin-X centrifuge tube (Costar, filter size 0.22 µM) and the samples were stored in −80°C until HPLC measurements. The samples from tissues were subjected to HPLC using a 20 mm × 3.9 mm Sentry Guard column (Nova-Pak C18 bonded silica) connected to a 150 mm × 4.6 mm Atlantis T3 silica-based, reversed-phase C18 columns (Waters Corporation). NAD⁺ and NAM were detected by UV detector and UV absorbance was monitored at 261 nm. Elution of NAD⁺ and NAM from samples was verified and quantified by co-elution with known amounts of NAD⁺ and NAM standards (Sigma-Aldrich).